Pancreatin is manufactured from porcine pancreatic tissue from animals that have been found suitable for human consumption after veterinary exams. Pancreatin is a mixture of digestive enzymes, mainly amylase, protease and lipase, extracted from porcine pancreas. Due to its important therapeutic properties and high level of safety, pancreatin has long been used as a pharmaceutical preparation in enzyme replacement therapy. A wide variety of pancreatin preparations are commercially available as a digestive enzyme supplement to aid digestion and enhance absorption of nutrients. Clinically, pancreatic enzyme replacement therapy is the mainstay of treatment for pancreatic exocrine insufficiency, which is associated with cystic fibrosis, chronic pancreatitis, post-gastrointestinal bypass surgery, post-pancreatectomy, etc.
A feature common to all biological products obtained from any material of animal or human origin is the risk of viral contamination. Biological contamination may arise either from the source material or from adventitious agents introduced during the manufacturing processes.
Viruses are small infectious agents that replicate only inside the living cells of other organisms. Viruses consist of nucleic acids (RNA or DNA) which are surrounded by a protein shell and in some case, a lipid layer. Viruses producing only a protein shell are commonly known as non-enveloped viruses and those with both protein and lipid components in the shell are commonly known as enveloped viruses. They range in size from about 15 nm to about 450 nm, and cannot be seen with light microscopes. The shape and structure of viruses has been studied by electron microscopy, NMR spectroscopy, and X-ray crystallography.
Viruses can infect all types of life forms, from animals and plants to bacteria and archaea. As viruses cannot replicate independently, they are reliant on hosts. Accordingly they occur in virtually all living things in the world. Very few of the known viruses are pathogenic for humans, as they are highly host specific. Several national authorities have urged pancreatin manufactures to improve the virus inactivation/removal capacities of the manufacturing processes; however, attempts at improvement have met with limited success, due to the fact that most conditions which would remove or inactivate viruses will also result in an inactive product (destroyed enzyme activities). Regulations and safety concerns mandate viral clearance (virus removal or inactivation) in biopharmaceuticals such as pancreatin active pharmaceutical ingredient (API). Consequently, companies producing pharmaceutical products derived from biological tissues are experiencing additional pressure from the regulatory bodies to increase the level of safety of their products by reducing all contaminants to the lowest level possible.
Some small non-enveloped DNA viruses such as porcine parvovirus (PPV) and porcine circovirus (PCV) are difficult to inactivate. Under conditions used to produce the commercial products, the PPV and PCV titers can be reduced but the viruses may not be completely inactivated. However, human infections by exposure to non-enveloped porcine viruses such as PPV and PCV from commercial products have never been reported despite widespread use of these porcine-derived commercial products in humans. For example, PCV DNAs were frequently detected in porcine-derived commercial product such as pepsin and a factor VIII concentrate; however the contaminated products could not elicit any infection when administrated intravenously into pigs [Fenaux et al. (2004), “A Chimeric Porcine Circovirus (PCV) with the Immunogenic Capsid Gene of the Pathogenic PCV Type 2 (PCV2) Cloned into the Genomic Backbone of the Nonpathogenic PCV1 Induces Protective Immunity against PCV2 Infection in Pigs”. J. Virology 78: 6297-6303]. PPV DNAs were detected in 21 of the 22 lots of Hyate: C porcine factor VIII concentrate, although serum samples from 98 Hyate:C human recipients all tested negative for PPV antibodies (Soucie et al. “Investigation of porcine parvovirus among persons with hemophilia receiving Hyate:C porcine factor VIII concentrate”. Tranfusion (2000) 40:708-711.). Giangrande et al. (“Viral pharmacovigilance study of haemophiliacs receiving porcine factor VIII”. Haemophilia (2002) 8:798-801.) also tested serum samples from 81 past recipients of porcine factor III and 125 other volunteers for evidence of antibodies against a range of porcine viruses including PPV, and the results were negative. Therefore, the risk of human infection by PPV, PCV or other non-enveloped porcine viruses that may still be present in commercial product is not supported by published reports, and such a risk, if it exists at all, is extremely small.
The present invention is directed to a method for reducing or inactivating viral and microbial content during a process for the manufacture of pancreatin without compromising purity, composition or potency as measured by enzymatic activity or enzyme activity ratios.
To date no reliable method has been developed for removal or inactivation of all viral contaminants in a pancreatin sample. This is due to the fact that the active enzymes in pancreatin are incompatible with many of the known inactivation conditions including heat, low pH, oxidation and ionizing irradiation. Nonetheless several methods have been published for the inactivation or reduction of viruses and microorganisms.
Tijssen et al (US 2014/0,017,223 A1) discloses a process for making a pancreatic enzyme preparation (PEP) comprising the step of reacting beta-propiolactone (BPL) with a preparation containing one or more pancreatic enzyme for a sufficient time to reduce a viral infectivity in the preparation.
Rämsch et al. (US patent 2011/0,268,844 A1) discloses pancreatin treated with high-pressure and or with a screen filtration followed by a high-pressure treatment of 4000, 5000 or 6000 bar for 5 minutes at 15° C. According to Rämsch, this is applicable to all virus forms, such as DNA and RNA viruses, enveloped and non-enveloped viruses and bacteria and fungi and comprising at least 50% of biological activity. He also further disclosed the unpredictability about whether inactivation of certain viruses using high-pressure treatment is actually successful. Different method condition must be selected depending on whether the samples are liquid or solid owing to the different compressibility of the samples.
Kurfurst, et al. (US 2009/0,233,344 A1) discloses a method for reducing viral and microbial contamination of a sample by treating the sample with a residual moisture of 0.5 weight % or less, subjecting the pancreatin treated with heat treatment at a temperature of 84° C., preferably 80° C. and below, for 48 hours or 30 hours, wherein the activity of the pancreatin obtained is at least 50% biological activity. The viral infectiousness of the pancreatin is disclosed as reduced by a log10 reduction factor of more than 1 log10.
Mann (US 2009/6,749,851) discloses methodology for sterilizing preparations of digestive enzymes to reduce the level of active biological contaminants such as viruses, bacteria, yeasts, molds, and fungi. The treatment of compositions comprising digestive enzymes involved stabilizing the compositions by either (a) reducing the temperature of (b) reducing the solvents of, or (c) adding a stabilizer to the composition, followed by irradiation of the composition.
Becher, et al. (US 2009/0,130,063 A1) discloses a process for separating an infectious viral load from a pancreatin sample for quantitatively determining the viral load in a pancreatin sample using centrifugation and ultra-centrifugation. This method has limitation for only liquid sample suitable for centrifugation.
Braeuniger et al. (Int. J. Hyg. Environ. Health (2000) 203: 71-75) discloses the use of heat for inactivation of the bovine parvovirus (BPV). It has been demonstrated BPV can be deactivated depending upon exposure to heat and residual moisture. However Braeuniger et al did not disclose any effects of heat on enzymatic activity and change in composition.
Lewis (US 1971/3,956,483) discloses a method of pancreatin processing and reduction of bacteria while maintaining the amylolytic, proteolytic and lipolytic activities. The method comprises heating the pancreatin to a sufficiently high temperature between 49-82° C. Lewis, however, fails to provide a process to inactivate or reduce the amount of viruses.
A particular challenge is the inactivation or reduction of viruses from a matrix of biological extracts whose active substance is enzyme mixtures, without destroying or changing the enzymatic activity or ratio of the proteins in the process. There is a demand for methods in which the viral and bacterial content in a biological extract which contains solids is reduced or minimized to the greatest possible extent consistent with preservation of the desired purity, composition and potency of the pharmaceutical active ingredient. The method must be equally suitable for solids and suspensions.
In accordance with ICH Q5A: “Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin”, the process developed herein required the reduction or inactivation of viruses from biological products while at the same time needed to maintain enzyme activities and compositional ratios (e.g. lipase, protease and amylase) at an acceptable level. In principle, pharmaceutically active agents should not contain infectious viruses. The current production processes are not able to reduce or inactivate potentially present non-enveloped viruses with a sufficient safety margin, and additional virus-reducing steps must be implemented.
PAA is considered an effective disinfectant that is able to inactivate a wide variety of bacteria (Cronmiller, J. R., et al. (1999), Efficacy of Conventional Endoscopic Disinfection and Sterilization Methods Against Helicobacter pylori Contamination. Helicobacter 4: 198-203.); fungi (Werner and Wewalka (1973), Oxidation of vitamin A alcohol with peracetic acid. Tetrahedron 29:47-50) and viruses (Kline and Hull (1960), The Virucidal Properties of Peracetic Acid. American Journal of Clinical Pathology 33: 30-33).
Peracetic acid (PAA) has been used also as a germicide in the spraying of fruits and vegetables (Greenspan and MacKeller (1951), “The Application of Peracetic Acid Germicidal Washes to Control Mold of Tomatoes (Food Technology 5:95-97).
Baldry (in J. Applied Bacteriology. (1983) 54:417-423; reported reduction by a factor of 106 in the number of vegetative bacteria within 1 min at 25° C. using a solution containing 1.3 mmol/L of PAA.
Hodde and Hiles (2002, Biotechnology and Bioengineering 79:211-216) demonstrated the use of PAA as a sterilant to inactivate several model viruses from porous, non-cross-linked, collagen-based material used for medical devices. Their work supports the safety of PAA treated materials for human use without fear of viral transmission.
Lomas et al. (in Cell and Tissue Banking (2004) 5: 149-160) reported that treatment of human Bone patellar tendon bone (BPTB) graft with PAA did not render cytotoxic or pro-inflammatory in vitro. BPTB grafts treated with PAA were more susceptible to collagenase degradation. They further described the high-level disinfection protocol, utilizing PAA and its positive effect on the biocompatibility and biomechanics of the patellar tendon allografts.